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A method is proposed for localization of the sites of affinity labelling of the p subunit of Escherichiu coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids.The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to shortterm treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the p subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-tcrminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed.The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the p subunit of E. coli RNA polymerase.Our earlier communications [l -91 have described highly selective affinity labelling of Escherichiu coli RNA polymerase according to the following scheme:where E is RNA polymerase, XpNl an affinity reagent which is an analogue of an initiating substrate and pp6N2 is a pyrimidine [a-"'PINTI'. Treatment according to this scheme is performed on the complex of the enzyme with a promoter. The high selectivity of the affinity labelling is due to t p fact that the covalent binding of the radioactive residue -pN2 at the second stage takes place under catalytic action of the center of phosphodiester bond synthesis of the enzyme. Recently the same approach has been successfully applied to RNA polymerase I1 of wheat germ [lo] and to RNA polymerase of T7 coliphage [Ill.In the case of E. coli RNA polymerase, treatment with a majority of reagents (in combination with a pyrimidine pp6N) resulted in selective labelling of the p subunit. Some of the targets of labelling were identified as Lys and His residuesThe amino acid sequence of the /I subunit is known [12]. We attempted to localize the sites of affinity labelling by traditional methods like complete trypsinolysis followed by isolation and sequencing of radioactive peptides, but these attempts have failed because of the small yield of affinity labelling. Therefore we developed a new method for the localization. The principle of this method has been reported briefly earlier [4, 6-9, 13, 141; it is based upon the random cleavage approach widely used for rapid sequencing of nucleic acids [15, 161 and involves random limited cleavage at methionine residues followed by gel electrophoresis of radioactive peptides...

RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [cx-~'P]NTP. Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNbN (where 5 = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive.Labelling took place only when the combination of the reagent and [U-~'P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short 'arms', only the fl subunit was labelled; the targets were His and/or Lys residues. With reagents having longer 'arms', the G subunit was also labelled.The value of information obtained by means of affinity modification of proteins (for review see [l]) depends on its selectivity, i.e. on the ratio of the extents of modification of amino acid residues inside and outside the ligand-binding center. It is difficult to achieve high selectivity with large enzymes and with analogues of substrates having moderate affinities to active centers. Escherichia coli RNA polymerase is a very large enzyme (relative molecular mass 500000) with moderate affinity to substrates '(Km z 0.01 mM). It is not surprising that numerous attempts to study its functional topography by means of affinity labelling with reactive analogues of substrates have given relatively poor results.Selectivity of affinity labelling may be increased by using differential labelling [2, 31, analogues of transition states [4, 51, and suicide substrates [6, 71. Certain basic disadvantages of the differential labelling technique make it practically inapplicable to RNA polymerase. As for the two other techniques, they depend upon availability of very specific reagents, and it is not yet clear what the structure of such reagents should be in the case of RNA polymerase.

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